tRNA m5U54 methyltransferase (RUMT), the trnA gene product, catalyzes the AdoMet-dependent methylation of uridine 54 in all E. coli tRNAs. Our group has shown that the mechanism of RUMT is similar to that of thymidylate synthase (TS). A nucleophile (C324) adds to C6 of U54 via Michael addition to form a RUMT-tRNA covalent adduct, activating C5 for direct attack on the incipient methyl of AdoMet. The stereochemistry of addition for RUMT proceeds via the cis route, differing from that of TS, which proceeds via the trans route. The minimal features of tRNA necessary for recognition are found within the T-arm of tRNA, and include the T-loop closed by at least two base pairs of the stem. By comparison of substrate tRNAs and extensive mutagenesis of the T-arm, a consensus sequence for substrate activity was constructed. These data suggest that RUMT may have other RNAs as substrates. In vitro selection (SELEX) experiments have been initiated to identify RNA sequences competent to bind RUMT. Such an approach may permit identification of non-tRNA RUMT substrates. The Computer Graphics Laboratory will be used to identify the structure of RUMT as well as to help to identify its catalytic residues by using information in the data bank.